The mechanism of pain in pancreatitis is poorly understood and its treatment remains difficult, in large part due to a lack of understanding of the underlying pathogenesis of nociceptor sensitization in this condition. Unlike that in other organs, inflammation in the pancreas is uniquely associated with a significant release of trypsin and other enzymes. Trypsin is an agonist of the protease activated receptor-2 (PAR-2), recently found to be expressed by nociceptor neurons and identified as a potentially key mediator of inflammatory hyperalgesia. In preliminary studies, using a specific PAR-2 activating peptide (AcPep), we found evidence of nociceptor sensitization in vitro in the form of enhanced capsaicin-and KC1-evoked release of CGRP, an important neuropeptide for nociceptive signaling. We then demonstrated that injection of AcPep into the pancreatic duct activated and sensitized pancreas-specific afferent neurons in vivo, as measured by spinal cord Fos expression. Further, we have shown that some of these effects can also be mimicked by activated trypsin. We therefore hypothesize that PAR-2 activation in the pancreas by trypsin activates and sensitizes the nociceptive response of this organ and contributes to the pain of pancreatitis. The overall goal of our studies is to characterize these effects further, using the following specific aims: 1) To study the effects of AcPep and trypsin on the pancreatic nociceptive response to painful stimuli and to determine if these are mediated by PAR-2. We will assess the response to intrapancreatic infusion of these PAR-2 agonists using in vivo behavioral and physiological assays of nociception in rats. The role of PAR-2 in mediating these effects will be tested by observing these responses, if any, in mice genetically engineered to lack PAR-2. 2) To study the effects of AcPep and trypsin on neurotransmitter release by primary afferent nociceptors and to determine if these are mediated by PAR-2. The effects of PAR-2 agonists on evoked CGRP release by cultured primary afferent neurons will be assessed and the role of PAR-2 in mediating these effects determined by performing similar studies on cultured neurons from PAR-2 knockout mice. 3) To determine the contribution PAR-2 to the overall nociceptive response in pancreatitis. The contribution of PAR-2 to pain in an experimental model of pancreatitis will be determined by behavioral, physiologic (spinal Fos expression) and neurotransmitter release studies in PAR-2 knockout mice. These studies will provide insight into the underlying mechanism of pancreatic pain that may eventually lead to new and more effective forms of therapy.